Dopamine (DA) is a key transmitter in motor, cognitive, and reward pathways of the brain, with dysfunction of DA transmission linked to significant disorders, including Parkinson's disease, schizophrenia, and addiction. The long-term goal of this project is to identify local factors that regulate somatodendritic DA release from DA neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) and axonal DA release in striatum. In the previous funding period, we focused on DA release regulation by endogenous glutamate, GABA, and Ca2+ entry, using real-time voltammetric recording of evoked DA release. We discovered that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is an intracellular messenger in SNc DA neurons that both modulates cell firing rate and inhibits somatodendritic DA release. By contrast, in dorsal striatum, H2O2 is a diffusible messenger that mediates regulation of axonal DA release by glutamate and GABA. These effects of H2O2 are mediated by the activation of ATP-sensitive K+ (KATP) channels. Proposed work will provide mechanistic insight into regulation of DA transmission by H2O2, as well as indicate functional consequences of H2O2 signaling on somatodendritic and axonal DA release. Aim 1 will test the hypothesis that H2O2 activates KATP channels by decreasing channel sensitivity to ATP;Aim 2 will determine the ionic dependence of H2O2 generation;Aim 3 will investigate the role of H2O2 generation in the regulation of somatodendritic DA release and DA cell physiology by glutamatergic NMDA receptors;and Aim 4 will evaluate the temporal and spatial characteristics of glutamate-dependent H2O2 signaling in dorsal striatum. Methods include voltammetric detection of DA release, whole-cell and excised patch recording, and fluorescence imaging of H2O2, intracellular ions, and mitochondrial membrane potential. Experimental systems include isolated DA neurons, transfected cells, and brain slices from guinea pigs and from mice lacking a specific KATP channel subtype. Several brain disorders that involve DA dysfunction, including Parkinson's disease and schizophrenia, have also been linked to oxidative stress. Proposed studies will clarify how endogenous H2O2 normally regulates DA release. Because unregulated H2O2 can lead to oxidative stress, however, the findings may also point to possible targets for therapeutic intervention in these debilitating disorders. This project is based on our novel finding that hydrogen peroxide is an endogenous factor that regulates the nigrostriatal dopamine pathway. Understanding factors that regulate this pathway is important, since it is nigrostriatal dopamine that is lost in Parkinson's disease, leaving individuals unable to move. Proposed studies will clarify how endogenous hydrogen peroxide normally regulates dopamine release and dopamine neuron activity in this pathway. Additionally, because unregulated peroxide can lead to oxidative stress, which is a causal factor in Parkinson's disease, the findings may also point to possible new targets for therapeutic intervention, consistent with one aspect of the mission of NINDS.